Plasmodium falciparum malaria exerts a devastating impact on public health in inter-tropical regions, causing 250 million cases and nearly one million deaths each year. The emergence and spread of chloroquine resistance (CQR) has decimated the efficacy of this former gold-standard antimalarial, and in SE Asia we are now witnessing the emergence of resistance to artemisinins, the fast-acting but very rapidly cleared core component of artemisinin-based combination therapies (ACTs). With precious little in the drug development pipeline, there is an imperative to understand mechanisms of antimalarial drug resistance as a means to optimize current treatments, detect resistance as it emerges, and provide knowledge and reagents to develop improved drugs. In this application, we focus on PfCRT, the primary determinant of P. falciparum CQR. In Aim 1 we test the hypothesis that the combination of intense CQ pressure and the need to sustain fitness in the face of competition with wild-type strains, has exerted competing selective forces on the evolution of mutant pfcrt that account for its considerable geographic diversity and complex repertoire of haplotypes. To study this, we propose to engineer mutant pfcrt alleles into CQ-sensitive P. falciparum strains from geographically matched regions, and study the impact of these alleles on the degree of CQR and the fitness of these mutant parasites in mixed infections with isogenic parasite lines expressing wild-type pfcrt. This will employ innovative zinc finger nuclease technology to rapidly and specifically target pfcrt for allelic exchange in any genetic background. These data can help predict where and how quickly CQ could regain efficacy and potentially be reintroduced, and identify partner drugs that counteract mutant pfcrt- mediated CQR. In Aim 2, we hypothesize that novel PfCRT mutations can arise that mediate high- level resistance to quinoline drugs used in first-line ACTs, with a focus on amodiaquine and piperaquine. The approach is based on allelic exchange approaches to examine recently discovered mutations, and the implementation of PCR-based random mutagenesis to select for novel mutations that impart high-level resistance. These data will provide molecular markers to screen for the emergence of resistance to these partner drugs, which essentially act as single agents once the artemisinin component has been cleared. In Aim 3, on the basis of exciting new data that provide mechanistic insights, we propose experiments to test the hypothesis that the native function of PfCRT involves metal-regulated redox sensing, and peptide transport that has been co-opted via the acquisition of point mutations to permit CQ efflux. This research, which is consistent with NIAID's mission statement, promises to provide significant new insights into PfCRT-mediated drug resistance, and inform strategies to detect resistant strains and identify improved treatments.